Post-transcriptional regulation has emerged as a potent way to control quantitatively and qualitatively gene expression. The RNase III family of double-stranded RNA endonucleases plays crucial functions in multiple gene expression pathways: they participate in the processing of precursors of ribosomal RNA, of small RNAs involved in splicing and rRNA metabolism, and of microRNAs. They also participate in the production of small interfering RNAs in the RNA interference process. Our long term goals are (i) to exhaustively identify the gene expression pathways controlled by eukaryotic members of the RNase III family of endonucleases, (ii) to understand the mechanisms by which these enzymes bind and cleave dsRNA and (iii) to understand how the activity of these enzymes is integrated in the cell metabolism. Using the S.cerevisiae enzyme Rntlp as a model eukaryotic RNase III enzyme and functional genomics, we will investigate novel gene expression pathways controlled by this enzyme. The functions of Rntlp in the surveillance and regulation of mRNAs encoding iron uptake proteins and for a protein involved in the methionine salvage pathway will be investigated. Novel genomic strategies will be developed to identify additional gene expression pathways regulated by eukaryotic RNases III. The recognition of double-stranded RNA and the mechanism of cleavage site selection by Rnt1p will be studied using site directed mutagenesis and site-specific modifications of the RNA substrate. The integration of RNase III activity in the cellular context will be investigated. In particular, we will decipher the mechanism by which RNase III is recruited cotranscriptionally to its substrates, and the mechanism by which RNase III is down-regulated in low iron conditions. These studies will contribute to our understanding of the roles of RNase III in the control of gene expression and of the mechanisms of adaptation of eukaryotic cell metabolism to iron deficiency.